The present invention relates to a novel physiologically active protein originating in mammals, a DNA encoding said protein, and an antibody reactive with said protein.
A so-called geriatric disease, which is regarded as a current disease in high living standard society, includes arteriosclerosis as well as hypertension and diabetes. Important measures for preventing these diseases are not only development of therapeutic methods but also daily life control.
Arteriosclerosis begins with pathological changes (for example, (1) invasive growth of smooth muscle cells into inner membrane, (2) qualitative and quantitative changes of collagen, elastin, and acidic mucopolysaccharides, and (3) cell foaming by lipid accumulation in the cytoplasm of grown smooth muscle cells and macrophages implanting tissues) occurring in inner membrane of artery. As the result of such pathological changes, (1) foam cells found in the inner membrane produces fat spots on the surface of the inner membrane, (2) lipid accumulates between tissues (deep part of midmembrane) and the inner membrane surface is covered with thick glass-like membrane, accompanied by fibrous growth and calcification, and (3) bleeding and necrosis occur in tissues to cause combined pathological changes involving thrombogenesis, calcification, and deposition of lipid crystals. Such pathological changes, in time, distribute in artery of a whole body and narrow the cavity of the artery. In addition, the site of pathological changes becomes bursal and the vascular wall loses elasticity, thereby hardening blood vessels. The vessels then wind, and normal blood flow is inhibited.
Epidemiological studies so far have illustrated age (about thirties or more), hypercholesterolemia, hypo-HDL-cholesterolemia, systolic hypertension, obesity, hemoglobin high value, and diabetes as risk factors of the onset of arteriosclerosis. Dynamics of in vivo factors inducing the onset include secretion of adrenalin, increase of thromboxane A2, decrease of prostacyclin, increase of serum peroxylipid, increase of free fatty acid, increase of platelet, increase of fibrinogen, increase of blood coagulation factors (XII and XIII), decrease of tissue plasmin, increase of prostaglandin, decrease of antithrombin III, increase of serum LDL, decrease of serum HDL, increase of insulin, and increase of renin.
Studies so far have revealed only that multiple conditions, for example, physical conditions such as age and obesity, complication with other diseases, and abnormalities of the dynamics of many in vivo factors complicatedly are related to each other to cause arteriosclerosis.
Treatments of arteriosclerosis are divided with their purpose into (1) preventive treatments to retract arteriosclerosis and to prevent the onset of arteriosclerosis by correcting lifestyle and physical abnormalities such as obesity (for example, diet therapy and therapeutic exercise) and (2) chemotherapy or surgical therapy to remove vessel occlusion symptoms occurring with the progress of arteriosclerosis or to prevent the onset of vessel cavity occlusion symptoms by thrombus or embolus,.
Since particular decisive causes of arteriosclerosis are unclear, only symptomatic treatment by chemotherapy is currently possible. For example, xcex2 blocker is applied when the enhancement of a catecholamine derivative such as adrenalin is suspected as the cause, eicosapentaenoic acid is applied for a prostaglandin derivative, vitamin E is applied for peroxylipid, and urokinase is applied for thrombus. No effective pharmaceuticals for treating the arteriosclerosis have been provided yet.
In the surgical therapy for arterial occulsion, percutaneous transluminal coronary angioplasty (PTCA) based on the observation by angiography prevails clinically as an effective means to enlarge vessel cavity. PTCA has remarkably progressed and prevailed since it was clinically applied by Gruntzig for the first time in 1977, and the number of the operation has rapidly increased in Japan.
PTCA is the method in which the occlusion (constriction) site is enlarged by inserting a thin catheter with a balloon at the tip in a thick catheter into the coronary artery occlusion site and by expanding the balloon.
However, in cavity enlargement by PTCA, restenosis occurs at the operation site of the artery in about 30 to 50% of the cases within a few months after the operation, and this restenosis is a major drawback of PTCA.
The restenosis has been thought to occur by the amplification of neonatal inner membrane proliferation based on the repair reaction of the injury site of the vascular wall, which has been inevitably caused by the enlargement of the occlusion site by PTCA. Although chemotherapy has been tried for preventing this restenosis, almost no effective drugs have been reported so far.
As mentioned above, at present, a method for the complete treatment and prevention of arteriosclerosis comprising the prevention of the recurrence of arteriosclerosis and the occurrence of restenosis has not established. It is thus desired to clarify the cause of the onset and progress of arteriosclerosis and to develop a method for the effective treatment and prevention thereof, and therapeutic and preventive drugs.
Coronary artery restenosis occurring after PTCA is regarded as a clinical model of arteriosclerosis from pathological viewpoints such as neonatal inner membrane proliferation or intimal thickening. Therefore, to diagnose the tissue characteristics of the vascular wall at the restenosis site after PTCA and to elucidate the difference between the characteristics and those of normal vascular wall by comparing them pathologically and at the gene level are effective to identify the cause and factors of restenosis, and further, arteriosclerosis.
In such comparative studies, a useful method for comparison and examination at the gene level using the genetic engineering technique is called differential display method (Nucleic Acids Research, Vol.21, No.18, pp.4272-4280 (1993); and Science, Vol.257, pp.967-971 (1992)).
Specifically, PCTA is applied to the coronary artery of a large mammal such as a rabbit, the expression patterns of genes in the inner membrane tissue at the PTCA site are examined by differential display method, and they are compared with the gene expression patterns in the inner membrane tissue without PCTA, to thereby identify genes specifically or increasingly expressed after PTCA.
Genes that express specifically or increasingly after PTCA and proteins derived from said genes may be closely related to arteriosclerosis and restenosis. The present invention provides pharmaceuticals and methods for preventing and treating arteriosclerosis and restenosis by identifying genes and proteins expressing specifically in arteriosclerosis and coronary artery restenosis.
As the result of studies on the analyses of genes specific to arteriosclerosis and/or coronary artery restenosis, the present inventors have discovered genes encoding two novel proteins (clone BA0306 and BA2303) that express increasingly at the comparatively early stage (day 1 to 7) after PTCA and completed the present invention.
The two novel protein-encoding genes of the present invention, whose characteristics are mentioned below, are expressed specifically after PTCA, and are thought to be genes involved in onset and progress of arteriosclerosis and/or coronary artery restenosis.
Clone BA0306 has the following characteristics.
(1) Its increased expression is observed on day 1 to 7 after
PTCA of coronary artery (the peak is observed on day 4).
(2) Northern blotting reveals the expression of the mRNA as about 3.5 k and about 4.4 k bands in various human tissues.
(3) It has ten putative transmembrane regions.
(4) It has amino acid sequence homology with S. cerevisiae oxidative stress resistance protein, S. cerevisiae zinc/cadmium resistance protein, heavy metal ion resistance protein, and so on.
(5) The molecules derived from humans and rabbits have the amino acid sequences of SEQ ID NO: 10 and 8, respectively. The molecule derived from mice has the amino acid sequence of SEQ ID NO: 28.
Judging from these characteristics, clone BA0306 is thought to inhibit active oxygen such as nitrogen monoxide (NO), which is involved in the progress of arteriosclerosis and/or restenosis.
Clone BA2303 has the following characteristics.
(1) Its increased expression is observed from day 1 after PTCA of coronary artery, and the expression continues until day 7 with the maximum expression on day 2 to 4.
(2) Northern blotting reveals the expression of the mRNA as about 3.9 k and about 2.1 k bands in various human tissues.
(3) It has seven putative transmembrane regions.
(4) The molecules derived from humans and mice have the amino acid sequences of SEQ ID NO: 4 and 6, respectively. The molecule derived from rabbits has the amino acid sequence of SEQ ID NO: 2.
Judging from these characteristics, clone BA2303 is thought to be a GTP binding protein (G protein)-coupled receptor that transmits a specific signal through intracellular G protein to an effector on the plasma membrane or the surface of the cytoplasm by binding to an in vivo ligand involved in the onset or progress of arteriosclerosis and/or restenosis.
Therefore, the genes (DNAs), proteins, or their fragments of the present invention and antibodies or a portion of them reactive with the proteins of the present invention are extremely useful for developing drugs for treatment and prevention of arteriosclerosis and for treatment and prevention of restenosis after PTCA for artery occlusion symptom and so on, targeting said genes or protein molecules. In addition, the DNAs of the present invention themselves are useful as antisense pharmaceuticals, extracellular region fragments of said proteins, for example, as soluble receptor pharmaceuticals, and said antibodies and a portion of them as antibody pharmaceuticals.
Genes (DNAs), proteins, and antibodies of the present invention are useful as reagents for searching proteins (ligands) interacting with the proteins of the present invention, thereby elucidating the function of said ligands, and developing therapeutic drugs targeting said ligands.
Based on the genetic information of the rabbit- or mouse-derived DNA, one embodiment of DNAs of the present invention, model animals (knockout animals) can be produced by disrupting (inactivating) the endogenous gene corresponding to the DNA. Similarly, transgenic animals can be produced as model animals by introducing the human-derived DNA, one embodiment of DNAs of the present invention, into nonhuman mammals such as mice. Function of genes and proteins of the present invention can be elucidated by analyzing the physical, biological, pathologic, and genetic characteristics of these model animals.
Moreover, by mating the model animals whose endogenous gene is thus disrupted with the transgenic animals, model animals that have only the human-derived gene of the present invention can be produced. By administering drugs (compounds, antibodies, and so on) targeting the introduced human gene to these model animals, the therapeutic effect of the drug can be estimated.
Namely, the present invention provides the DNAs, proteins, expression vectors, transformants, antibodies, pharmaceutical compositions, transgenic mice, and knockout, mentioned below.
(1) A DNA encoding a protein having the amino acid sequence of SEQ ID NO: 4.
(2) A DNA encoding a protein fragment comprising the extracellular region of a protein having the amino acid sequence of SEQ ID NO: 4.
(3) A DNA comprising a nucleotide sequence corresponding to nucleotide residues 97 to 1419 of the nucleotide sequence of SEQ ID NO: 3.
(4) A DNA hybridizing with a DNA having the nucleotide sequence of SEQ ID NO: 3 under stringent conditions.
(5) A protein having the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence substantially the same as said amino acid sequence.
(6) A protein fragment comprising the extracellular region of a protein having the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence substantially the same as said amino acid sequence.
(7) A fusion protein between the extracellular region of the protein of (5) and the constant region of the heavy chain of human immunoglobulin (Ig) or a portion of the constant region.
(8) An expression vector comprising the DNA of any one of (1) to (4).
(9) A transformant carrying the expression vector of (8).
(10) An antibody or its portion reactive with the protein of (5) or the protein fragment of (6).
(11) The antibody or its portion of (10), wherein the antibody is a monoclonal antibody.
(12) A pharmaceutical composition comprising the protein fragment of (6) or the fusion protein of (7) and a pharmaceutically acceptable carrier.
(13) A pharmaceutical composition comprising the antibody or its portion of (10) or (11) and a pharmaceutically acceptable carrier.
(14) A DNA encoding a protein having the amino acid sequence of SEQ ID NO: 10.
(15) A DNA encoding a protein fragment comprising the extracellular region of a protein having the amino acid sequence of SEQ ID NO: 10.
(16) A DNA having a nucleotide sequence corresponding to nucleotide residues 1 to 1785 of the nucleotide sequence of SEQ ID NO: 9.
(17) A DNA hybridizing with a DNA having the nucleotide sequence of SEQ ID NO: 9 under stringent conditions.
(18) A protein having the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence substantially the same as said amino acid sequence.
(19) A protein fragment comprising the extracellular region of a protein having the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence substantially the same as said amino acid sequence.
(20) A fusion protein comprising the extracellular region of the protein of (18) and the constant region of the heavy chain of human immunoglobulin (Ig) or a portion of the constant region.
(21) An expression vector comprising the DNA of any one of (14) to (17).
(22) A transformant carrying the expression vector of (21).
(23) An antibody or its portion reactive with the protein of (18) or the protein fragment of (19).
(24) The antibody or its portion of (23), wherein the antibody is a monoclonal antibody.
(25) A pharmaceutical composition comprising the protein fragment of (19) or the fusion protein of (20) and a pharmaceutically acceptable carrier.
(26) A pharmaceutical composition comprising the antibody or its portion of (23) or (24) and a pharmaceutically acceptable carrier.
(27) A transgenic mouse in which the human-derived DNA comprising a DNA having a nucleotide sequence corresponding to nucleotide residues 97 to 1419 of the nucleotide sequence of SEQ ID NO: 3 is integrated into an endogenous gene of said mouse.
(28) A transgenic mouse in which the human-derived DNA comprising a DNA having a nucleotide sequence corresponding to nucleotide residues 1 to 1785 of the nucleotide sequence of SEQ ID NO: 9 is integrated into an endogenous gene of said mouse.
(29) A knockout mouse whose endogenous gene encoding a mouse-derived protein having the amino acid sequence of SEQ ID NO: 6 is inactivated so that said protein is not produced.
(30) A knockout mouse whose endogenous gene encoding a mouse-derived protein comprising the amino acid sequence of SEQ ID NO: 28 is inactivated so that said protein is not produced.
In the following, the present invention is explained in detail by clarifying the meanings of terms used in the present application and the general production methods of proteins, protein fragments, fusion proteins, DNAS, antibodies, transgenic mice, and knockout mice of the present invention.
A xe2x80x9cproteinxe2x80x9d of the present invention means a protein and its fragment derived from mammals such as humans, rabbits, and mice, and preferably, a human-derived protein and its fragment. Particularly preferable examples are (1) a protein having the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence substantially the same as said amino acid sequence, (2) a protein fragment comprising the extracellular region of a protein having the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence substantially the same as said amino acid sequence, (3) a protein having the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence substantially the same as said amino acid sequence, and (4) a protein fragment comprising the extracellular region of a protein having the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence substantially the same as said amino acid sequence.
The term xe2x80x9cextracellular regionxe2x80x9d used herein is explained below. A transmembrane protein such as a G protein-coupled receptors or cell surface molecule connects with the membrane through the hydrophobic peptide region penetrating the lipid bilayer of the membrane once or several times and has structure composed of three main regions, that is, extracellular region, transmembrane region, and cytoplasmic region. Such a transmembrane protein exists as a- monomer, homodimer, heterodimer, or oligomer with another chain(s) having the same or different amino acid sequence.
The term xe2x80x9cextracellular regionxe2x80x9d used herein means the partial structure (partial sequence) existing outside of the membrane that holds the transmembrane protein as mentioned above among the whole structure of said membrane protein. In other words, it corresponds to the region excluding the region incorporated into the membrane (transmembrane region) and the region existing in the cytoplasm following the transmembrane region (cytoplasmic region). If desired, one to five amino acids derived from the amino acids constituting the transmembrane and/or cytoplasmic region can be added to the N- terminus and/or C-terminus of the extracellular region in the present invention.
Here, xe2x80x9chaving substantially the same amino acid sequencexe2x80x9d means to include a protein having an amino acid sequence where multiple amino acids, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids, in the amino acid sequence shown in SEQ ID NO: 4 or 10, are substituted, deleted, and/or modified, and a protein having an amino acid sequence where multiple amino acids, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids, are added to said amino acid sequence, as far as the protein has substantially the same biological properties as the protein having said amino acid sequence.
Alphabetical triplet or single letter codes used to represent amino acids in the present specification or figures mean amino acids as follows. (Gly/G) glycine, (Ala/A) alanine, (Val/V) valine, (Leu/L) leucine, (Ile/I) isoleucine, (Ser/S) serine, (Thr/T) threonine, (Asp/D) aspartic acid, (Glu/E) glutamic acid, (Asn/N) asparagine, (Gln/Q) glutamine, (Lys/K) lysine, (Arg/R) arginine, (Cys/C) cysteine, (Met/M) methionine, (Phe/F) phenylalanine, (Tyr/Y) tyrosine, (Trp/W) tryptophane, (His/H) histidine, (Pro/P) proline.
xe2x80x9cThe constant region or a portion of the constant region of human immunoglobulin (Ig) heavy chainxe2x80x9d used herein means the constant region or the Fc region of human-derived immunoglobulin heavy chain (H chain) as described, or a portion of them. The immunoglobulin can be any immunoglobulin belonging to any class and any subclass. Specifically, examples of the immunoglobulin are IgG (IgG1, IgG2, IgG3, and IgG4), IgM, IgA (IgA1 and IgA2), IgD, and IgE. Preferably, the immunoglobulin is IgG (IgG1, IgG2, IgG3, or IgG4), or IgM. Examples of particularly preferable immunoglobulin of the present invention are those belonging to human-derived IgG (IgG1, IgG2, IgG3, or IgG4).
Immunoglobulin has a Y-shaped structural unit in which four chains composed of two homologous light chains (L chains) and two homologous heavy chains (H chains) are connected through disulfide bonds (Sxe2x80x94S bonds). The light chain is composed of the light chain variable region (VL) and the light chain constant region (CL). The heavy chain is composed of the heavy chain variable region (VH) and the heavy chain constant region (CH).
The heavy chain constant region is composed of some domains having the amino acid sequences inherent in each class (IgG, IgM, IgA, IgD, and IgE) and each subclass (IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2).
The heavy chain of IgG (IgG1, IgG2, IgG3, and IgG4) is composed of VH, CH1 domain, hinge region, CH2 domain, and CH3 domain in this order from N terminus.
Similarly, the heavy chain of IgG1 is composed of VH, Cxcex311 domain, hinge region, Cxcex312 domain, and Cxcex313 domain in this order from N terminus. The heavy chain of IgG2 is composed of VH, Cxcex321 domain, hinge region, Cxcex322 domain, and Cxcex323 domain in this order from N terminus. The heavy chain of IgG3 is composed of VH, Cxcex331 domain, hinge region, Cxcex332 domain, and Cxcex333 domain in this order from N terminus. The heavy chain of IgG4 is composed of VH, Cxcex341 domain, hinge region, Cxcex342 domain, and Cxcex343 domain in this order from N terminus.
The heavy chain of IgA is composed of VH, Cxcex11 domain, hinge region, Cxcex12 domain, and Cxcex13 domain in this order from N terminus.
Similarly, the heavy chain of IgA1 is composed of VH, Cxcex111 domain, hinge region, Cxcex112 domain, and Cxcex113 domain in this order from N terminus. The heavy chain of IgA2 is composed of VH, Cxcex121 domain, hinge region, Cxcex122 domain, and Cxcex123 domain in this order from N terminus.
The heavy chain of IgD is composed of VH, Cxcex41 domain, hinge region, Cxcex42 domain, and Cxcex43 domain in this order from N terminus.
The heavy chain of IgM is composed of VH, Cxcexc1 domain, Cxcexc2 domain, Cxcexc3 domain, and Cxcexc4 domain in this order from N terminus and have no hinge region as seen in IgG, IgA, and IgD.
The heavy chain of IgE is composed of VH, Cxcex51 domain, Cxcex52 domain, Cxcex53 domain, and Cxcex54 domain in this order from N terminus and have no hinge region as seen in IgG, IgA, and IgD.
If, for example, IgG is treated with papain, it is cleaved at the slightly N terminal side beyond the disulfide bonds existing in the hinge region where the disulfide bonds connect the two heavy chains to generate two homologous Fab, in which a heavy chain fragment composed of VH and CH1 is connected with one light chain through a disulfide bond, and one Fc, in which two homologous heavy chain fragments composed of the hinge region, CH2 domain, and CH3 domain are connected through disulfide bonds (See xe2x80x9cImmunology Illustratedxe2x80x9d, original 2nd ed., Nankodo, pp.65-75 (1992); and xe2x80x9cFocus of Newest Medical Science xe2x80x98Recognition Mechanism of Immune Systemxe2x80x99xe2x80x9d, Nankodo, pp.4-7 (1991); and so on).
Namely, xe2x80x9ca portion of a constant region of immunoglobulin heavy chainxe2x80x9d of the present invention means a portion of a constant region of an immunoglobulin heavy chain having the structural characteristics as mentioned above, and preferably, is the constant region without C1 domain, or the Fc region. Specifically, examples thereof are the region composed of hinge region, C2 domain, and C3 domain from each of IgG, IgA, and IgD, and are the region composed of C2 domain, C3 domain, and C4 domain from each of IgM and IgE. A particularly preferable example thereof is the Fc region of human-derived IgG1.
The xe2x80x9cfusion proteinxe2x80x9d of the present invention is that composed of the above-described extracellular region of the protein of the present invention and a constant region or a portion of a constant region of human immunoglobulin (Ig) heavy chain. Preferably, it is a fusion polypeptide composed of an extracellular region of a protein of the present invention and a portion of a constant region of human IgG heavy chain, and particularly preferably, it is a fusion polypeptide composed of an extracellular region of a protein of the present invention and the region (Fc) composed of a hinge region, CH2 domain, and CH3 domain of human IgG heavy chain. Moreover, IgG1 is preferable among IgG. In addition, a protein derived from human, mouse, or rat (preferably, human) is preferable as the protein of the present invention.
The fusion protein of the present invention has the advantage that the fusion polypeptide can be purified extremely easily by using affinity column chromatography using the property of protein A, which binds specifically to the immunoglobulin fragment because the fusion polypeptide of the present invention has a portion of a constant region (for example Fc) of an immunoglobulin such as IgG as mentioned above as a fusion partner. Moreover, since various antibodies against the Fc of various immunoglobulin are available, an immunoassay for the fusion polypeptides can be easily performed with antibodies against the Fc.
The protein, protein fragment, and fusion protein of the present invention can be produced not only by recombinant DNA technology as mentioned below but also by a method well known in the art such as a chemical synthetic method and a cell culture method, or a modified method thereof.
The DNA of the present invention encodes the above-mentioned protein of the present invention, and includes any nucleotide sequence that can encode the protein of the present invention. The DNA preferably encodes a human-derived protein of the present invention. Specific examples of the DNA are described below.
(1) A DNA encoding a protein having the amino acid sequence of SEQ ID NO: 4, a protein fragment composed of the extracellular region of said protein, or a biological analog obtained by substituting, deleting, and/or modifying multiple amino acids, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids in the amino acid sequence of said protein or fragment, or by inserting multiple amino acids, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids, in said amino acid sequence.
(2) A DNA encoding a protein having the amino acid sequence of SEQ ID NO: 10, a protein fragment composed of the extracellular region of said protein, or a biological analog obtained by substituting, deleting, and/or modifying multiple amino acids, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids, in the amino acid sequence of said protein or fragment, or by inserting multiple amino acids, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids, in said amino acid sequence.
(3) A DNA hybridizing with a DNA having the nucleotide sequence of SEQ ID NO: 3 under stringent conditions.
(4) A DNA hybridizing with a DNA having the nucleotide sequence of SEQ ID NO: 9 under stringent conditions.
Specific examples thereof are (1) a DNA having a nucleotide sequence corresponding to nucleotide residues 97 to 1419 of the nucleotide sequence of SEQ ID NO: 3, (2) a DNA comprising a nucleotide sequence corresponding to nucleotide residues 1 to 1419 of the nucleotide sequence of SEQ ID NO: 3, (3) a DNA having a nucleotide sequence corresponding to nucleotide residues 1 to 1785 of the nucleotide sequence of SEQ ID NO: 9, and (4) a DNA comprising a nucleotide sequence corresponding to nucleotide residues 1 to 1785 of the nucleotide sequence of SEQ ID NO: 9.
The DNA of the present invention comprises either a genomic DNA or cDNA. In addition, the DNA includes any DNA composed of any codons encoding the same amino acids.
Examples of xe2x80x9cstringent conditionsxe2x80x9d are as follows. When a probe with 50 or more nucleotides is used and hybridization is performed in 0.9% NaCl, the standard of temperature where 50% dissociation occurs (Tm) is calculated using the following formula and the temperature for hybridization can be determined according to the following formula.
Tm=82.3xc2x0 C.+0.41xc3x97(G+C)%xe2x88x92500/nxe2x88x920.61xc3x97(formamide)%
(n means the number of the nucleotide of probe).
Temperature=Tm xe2x88x9225xc2x0 C.
In addition, when a probe with 100 or more nucleotides (G+C=40 to 50%) is used, it should be considered that Tm varies as (1) and (2) mentioned below.
(1) Tm descends by about 1xc2x0 C. per 1% mismatch.
(2) Tm descends by 0.6 to 0.7xc2x0 C. per 1% formamide.
Accordingly, the temperature conditions for the combination of completely complementary strands can be set as follows.
(A) 65 to 75xc2x0 C. (formamide not added)
(B) 35 to 45xc2x0 C. (in the presence of 50% formamide)
The temperature conditions for the combination of incompletely complementary strands can be set as follows.
(A) 45 to 55xc2x0 C. (formamide not added)
(B) 35 to 42xc2x0 C. (in the presence of 30% formamide)
The temperature conditions when a probe with 23 or less nucleotides is used can be 37cC or can be calculated using the following formula.
Temperature=2xc2x0 C.xc3x97(the number of A+T)+4xc2x0 C.xc3x97(the number of C+G)xe2x88x925xc2x0 C.
The DNA of the present invention can be a DNA obtained by any method. For example, the DNA includes complementary DNA (cDNA) prepared from mRNA, DNA prepared from genomic DNA, DNA prepared by chemical synthesis, DNA obtained by PCR amplification with RNA or DNA as a template, and DNA constructed by appropriately combining these methods.
The DNA encoding the protein of the present invention can be obtained by the usual method such as a method to clone cDNA from mRNA encoding the protein of the present invention, a method to isolate genomic DNA and then splice them, chemical synthesis and so on.
(1) cDNA can be cloned from the mRNA encoding the protein of the present invention by, for example, the method described below.
First, the mRNA encoding the protein of the present invention is prepared from the above-described tissues or cells expressing and producing a cell surface molecule (polypeptide) of the present invention. mRNA can be prepared isolating total RNA by a known method such as quanidine-thiocyanate method (Chirgwin et al., Biochemistry, Vol.18, p5294, 1979), hot phenol method, or AGPC method, and subjecting it to affinity chromatography using oligo-dT cellulose or poly-U Sepharose.
Then, with the mRNA obtained as a template, cDNA is synthesized, for example, by a well-known method using reverse transcriptase such as the method of Okayama et al. (Mol. Cell. Biol. Vol.2, p.161 (1982); ibid. Vol.3, p.280 (1983)) or the method of Hoffman et al. (Gene Vol.25, p.263 (1983)), and converted into double-stranded cDNA. A cDNA library is prepared by transforming E. coli with plasmid vectors, phage vectors, or cosmid vectors having this cDNA or by transfecting E. coli after in vitro packaging.
The plasmid vectors used in this invention are not limited as long as they are replicated and maintained in hosts. Any phage vectors that can be replicated in hosts can also be used. Examples of usually used cloning vectors are pUC19, xcexgt10, xcexgt11, and so on. When the vector is applied to immunological screening as mentioned below, the vector having a promoter that can express a gene encoding the polypeptide of the present invention in a host is preferably used.
cDNA can be inserted into a plasmid by, for example, the method of Maniatis et al. (Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory, p.1.53, 1989). cDNA can be inserted into a phage vector by, for example, the method of Hyunh et al. (DNA cloning, a practical approach, Vol.1, p.49 (1985)). These methods can be simply performed by using a commercially available cloning kit (for example, a product from Takara Shuzo). The recombinant plasmid or phage vector thus obtained is introduced into appropriate host cells such as a prokaryote (for example, E. coli: HB101, DH5xcex1, MC1061/P3, etc.).
Examples of a method for introducing a plasmid into a host are calcium chloride method, calcium chloride/rubidium chloride method described in Molecular Cloning, A Laboratory Manual (second edition, Cold Spring Harbor Laboratory, p.1.74 (1989)), and electroporation method. Phage vectors can be introduced into host cells by, for example, a method in which the phage DNAs are introduced into grown hosts after in vitro packaging. In vitro packaging can be easily performed with a commercially available in vitro packaging kit (for example, a product from Stratagene or Amersham).
The cDNA encoding the protein of the present invention can be isolated from the cDNA library so prepared according to the method mentioned above by combining general cDNA screening methods.
For example, a clone comprising the desired cDNA can be screened by a known colony hybridization method (Crunstein et al. Proc. Natl. Acad. Sci. USA, Vol.72, p.3961 (1975)) or plaque hybridization method (Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory, p.2.108 (1989)) using 32P-labeled chemically synthesized oligonucleotides as probes, which are corresponding to the amino acid sequence of the polypeptide of the present invention. Alternatively, a clone having a DNA fragment encoding a specific region within the polypeptide of the present invention can be screened by amplifying the region by PCR with synthetic PCR primers.
When a cDNA library prepared using a cDNA expression vector (for example, xcexZAPII phage vector) is used, the desired clone can be screened by the antigen-antibody reaction using an antibody against the polypeptide of the present invention. A screening method using PCR method is preferably used when many clones are subjected to screening.
The nucleotide sequence of the DNA thus obtained can be determined by Maxam-Gilbert method (Maxam et al. Proc. Natl. Acad. Sci. USA, Vol.74, p.560 (1977)) or the dideoxynucleotide synthetic chain termination method using phage M13 (Sanger et al. Proc. Natl. Acad. Sci. USA, Vol.74, pp.5463-5467 (1977)). The whole or a portion of the gene encoding the polypeptide of the present invention can be obtained by excising the clone obtained as mentioned above with restriction enzymes and so on.
(2) The DNA encoding the polypeptide of the present invention can be isolated from the genomic DNA derived from the cells expressing the polypeptide of the present invention as mentioned above by the following methods. Such cells are solubilized preferably by SDS or proteinase K, and the DNAs are deproteinized by repeating phenol extraction. RNAs are digested preferably with ribonuclease. The DNAs obtained are partially digested with appropriate restriction enzymes, and the DNA fragments obtained are amplified with appropriate phage or cosmid to generate a library. Then, clones having the desired sequence are detected, for example, by using radioactively labeled DNA probes, and the whole or a portion of the gene encoding the protein of the present invention is obtained from the clones by excision with restriction enzyme and so on.
(3) The DNA of the present invention can also be chemically synthesized by the usual method, based on the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, or 27.
The present invention also relates to a recombinant vector comprising the DNA encoding the protein of the present invention. The recombinant vector of the present invention is not limited as long as it can be replicated and maintained or can autonomously replicate in various prokaryotic and/or eukaryotic hosts. The vector of the present invention includes plasmid vectors and phage vectors.
The recombinant vector can easily be prepared by ligating the DNA encoding the protein of the present invention with a vector for recombination available in the art (plasmid DNA and bacteriophage DNA) by the usual method. Specific examples of the vectors for recombination used are E. coli-derived plasmids such as pBR322, pBR325, pUC12, pUC13, and pUC19, yeast-derived plasmids such as pSH19 and pSH15, and Bacillus subtilis-derived plasmids such as pUB110, pTP5, and pC194. Examples of phages are a bacteriophage such as xcex phage, and an animal or insect virus (pVL1393, Invitrogen) such as a retrovirus, vaccinia virus, and nuclear polyhedrosis virus.
An expression vector is useful for expressing the DNA encoding the protein of the present invention and for producing the polypeptide of the present invention. The expression vector is not limited as long as it expresses the gene encoding the polypeptide of the present invention in various prokaryotic and/or eukaryotic host cells and produces this protein. Examples thereof are pMAL C2, pEF-BOS (Nucleic Acids Res. Vol.18, p.5322 (1990)), pME18S (Experimental Medicine: SUPPLEMENT, xe2x80x9cHandbook of Genetic Engineeringxe2x80x9d (1992)), and so on.
When bacteria, particularly E. coli are used as host cells, an expression vector is generally comprised of, at least, a promoter/operator region, an initiation codon, the DNA encoding the protein of the present invention, termination codon, terminator region, and replicon.
When yeast, animal cells, or insect cells are used as hosts, an expression vector is preferably comprised of, at least, a promoter, an initiation codon, the DNA encoding the protein of the present invention, and a termination codon. It may also comprise the DNA encoding a signal peptide, enhancer sequence, 5xe2x80x2- and 3xe2x80x2-untranslated region of the gene encoding the protein of the present invention, splicing junctions, polyadenylation site, selectable marker region, and replicon. The expression vector may also contain, if required, a gene for gene amplification (marker) that is usually used.
A promoter/operator region to express the polypeptide of the present invention in bacteria comprises a promoter, an operator, and a Shine-Dalgarno (SD) sequence (for example, AAGG). For example, when the host is Escherichia, it preferably comprises Trp promoter, lac promoter, recA promoter, xcexPL promoter, lpp promoter, tac promoter, or the like. Examples of a promoter to express the polypeptide of the present invention in yeast are PH05 promoter, PGK promoter, GAP promoter, ADH promoter, and so on. When the host is Bacillus, examples thereof are SL01 promoter, SP02 promoter, penP promoter and so on. When the host is a eukaryotic cell such as a mammalian cell, examples thereof are SV40-derived promoter, retrovirus promoter, heat shock promoter, and so on, and preferably SV-40 and retrovirus-derived one. As a matter of course, the promoter is not limited to the above examples. In addition, to use an enhancer is effective for expression.
A preferable initiation codon is, for example, a methionine codon (ATG).
The commonly used termination codon (for example, TAG, TGA, TAA, and so on) is illustrated as a termination codon.
Usually used natural or synthetic terminators are used as a terminator region.
A replicon means a DNA capable of replicating the whole DNA sequence in host cells, and includes a natural plasmid, an artificially modified plasmid (DNA fragment prepared from a natural plasmid), a synthetic plasmid, and so on. Examples of a preferable plasmids are pBR322 or its artificial derivatives (DNA fragment obtained by treating pBR322 with appropriate restriction enzymes) forE. coli, yeast 2xcexc plasmid or yeast chromosomal DNA for yeast, and pRSVneo ATCC 37198, pSV2dhfr ATCC 37145, pdBPV-MMTneo ATCC 37224, pSV2neo ATCC 37149, etc. for mammalian cells.
An enhancer sequence, polyadenylation site, and splicing junction that are usually used in the art, such as those derived from SV40 can be also used.
A selectable marker usually used can be used according to the usual method. Examples thereof are resistance genes for antibiotics, such as tetracycline, neomycin, ampicillin, or kanamycin, and thymidine kinase gene.
Examples of a gene for gene amplification are dihydrofolate reductase (DHFR) gene, thymidine kinase gene, neomycin resistance gene, glutamate synthase gene, adenosine deaminase gene, ornithine decarboxylase gene, hygromycin-B-phophotransferase gene, aspartate transcarbamylase gene, etc.
The expression vector of the present invention can be prepared by continuously and circularly linking at least the above-mentioned promoter, initiation codon, DNA (gene) encoding the polypeptide of the present invention, termination codon, and terminator region, to an appropriate replicon. If desired, appropriate DNA fragments (for example, linkers, restriction sites generated with other restriction enzyme), can be used by the usual method such as digestion with a restriction enzyme or ligation using T4 DNA ligase.
Transformants of the present invention can be prepared by introducing the expression vector mentioned above into host cells.
Host cells used in the present invention are not limited as long as they are compatible with an expression vector mentioned above and can be transformed. Examples thereof are various cells such as natural cells or artificially established recombinant cells usually used in technical field of the present invention (for example, bacteria (Escherichia and Bacillus), yeast (Saccharomyces, Pichia, etc.), animal cells, or insect cells.
E. coli or animal cells are preferably used. Specific examples are E. coli (DH5xcex1, TB1, HB101, etc.), mouse-derived cells (COP, L, C127, Sp2/0, NS-1, NIH 3T3, etc.), rat-derived cells, hamster-derived cells (BHK, CHO, etc.), monkey-derived cells (COS1, COS3, COS7, CV1, Velo, etc.), and human-derived cells (Hela, diploid fibroblast-derived cells, HEK293, myeloma, Namalwa, etc.).
An expression vector can be introduced (transformed (transduced)) into host cells by known method.
Transformation can be performed, for example, according to the method of Cohen et al. (Proc. Natl. Acad. Sci. USA, Vol.69, p.2110 (1972)), protoplast method (Mol. Gen. Genet., Vol.168, p.111 (1979)), or competent method (J. Mol. Biol., Vol.56, p.209 (1971)) when the hosts are bacteria (E. coli, Bacillus subtilis, etc.), the method of Hinnen et al. (Proc. Natl. Acad. Sci. USA, Vol.75, p.1927 (1978)), or lithium method (J. Bacteriol., Vol.153, p.163 (1983)) when the host is Saccharomyces cerevisiae, the method of Graham (Virology, Vol.52, p.456 (1973)) when the hosts are animal cells, and the method of Summers et al. (Mol. Cell. Biol., Vol.3, pp.2156-2165 (1983)) when the hosts are insect cells.
The protein of the present invention can be produced by cultivating transformants (in the following this term includes transductants) comprising an expression vector prepared as mentioned above in nutrient media.
The nutrient media preferably comprise carbon source, inorganic nitrogen source, or organic nitrogen source necessary for the growth of host cells (transformants). Examples of the carbon source are glucose, dextran, soluble starch, and sucrose, and examples of the inorganic or organic nitrogen source are ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meet extract, soy bean cake, and potato extract. If desired, they may comprise other nutrients (for example, an inorganic salt (for example, calcium chloride, sodium dihydrogenphosphate, and magnesium chloride), vitamins, antibiotics (for example, tetracycline, neomycin, ampicillin, kanamycin, etc.).
Cultivation is performed by a method known in the art. Cultivation conditions such as temperature, pH of the media, and cultivation time are selected appropriately so that the protein of the present invention is overproduced.
Specific media and cultivation conditions used depending on host cells are illustrated below, but are not limited thereto.
When the hosts are bacteria, actinomycetes, yeasts, filamentous fungi, liquid media comprising the nutrient source mentioned above are appropriate. The media with pH 5 to 8 are preferably used.
When the host is E. coli, examples of preferable media are LB media, and M9 media (Miller et al. Exp. Mol. Genet., Cold Spring Harbor Laboratory, p.431 (1972)). Using these media, cultivation can be performed usually at 14 to 43xc2x0 C. for about 3 to 24 hours with aeration and stirring, if necessary.
When the host is Bacillus, cultivation can be performed usually at 30 to 40xc2x0 C. for about 16 to 96 hours with aeration and stirring, if necessary.
When the host is yeast, examples of media are Burkholder minimal media (Bostian, Proc. Natl. Acad. Sci. USA, Vol.77, p.4505 (1980)). The pH of the media is preferably 5 to 8. Cultivation can be performed usually at 20 to 35xc2x0 C. for about 14 to 144 hours with aeration and stirring, if necessary.
When the host is an animal cell, examples of media are MEM media containing about 5 to 20% fetal bovine serum (Science, Vol.122, p.501 (1952)), DMEM media (Virology, Vol.8, p.396 (1959)), RPMI1640 media (J. Am. Med. Assoc., Vol.199, p.519 (1967)), and 199 media (Proc. Soc. Exp. Biol. Med., Vol.73, p.1 (1950)). The pH of the media is preferably about 6 to 8. Cultivation can be performed usually at about 30 to 40xc2x0 C. for about 15 to 72 hours with aeration and stirring, if necessary.
When the host is an insect cell, an example of media is Grace""s media containing fetal bovine serum (Proc. Natl. Acad. Sci. USA, Vol.82, p.8404 (1985)). The pH thereof is preferably about 5to 8. Cultivation can be performed usually at about 20 to 40xc2x0 C. for 15 to 100 hours with aeration and stirring, if necessary.
The protein of the present invention can be produced as a transmembrane protein by cultivating transformants as mentioned above, in particular animal cells to overexpress the protein of the present invention on the surface of the cells. The protein of the present invention can be produced as a soluble protein fragment such as an extracellular region protein fragment by preparing the transformants as mentioned above using the DNA encoding the extracellular region and by cultivating the transformants to allow them to secrete the soluble polypeptide into the culture supernatant.
Namely, a culture filtrate (supernatant) is obtained by the method such as filtration or centrifugation of the obtained culture, and the protein of the present invention is purified and isolated from the culture filtrate by the usual method commonly used in order to purify and isolate a natural or synthetic protein.
Examples of the isolation and purification method are a method utilizing solubility, such as salting out and solvent precipitation method, a method utilizing the difference in molecular weight, such as dialysis, ultrafiltration, gel filtration, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a method utilizing charges, such as ion exchange chromatography and hydroxylapatite chromatography, a method utilizing specific affinity, such as affinity chromatography, a method utilizing the difference in hydrophobicity, such as reverse phase high performance liquid chromatography, and a method utilizing the difference in isoelectric point, such as isoelectric focusing.
When the protein of the present invention exists in the periplasm or cytoplasm of cultured transformants, first, the fungus bodies or cells are harvested by the usual method such as filtration or centrifugation and suspended in appropriate buffer. After the cell wall and/or cell membrane of the cells and so on are disrupted by the method such as lysis with sonication, lysozyme, and freeze-thawing, the membrane fraction comprising the protein of the present invention is obtained by the method such as centrifugation or filtration. The membrane fraction is solubilized with a detergent such as Triton-X100 to obtain the crude extract. Finally, the polypeptide or the polypeptide fragment is isolated and purified from the crude extract by the usual method as illustrated above.
The xe2x80x9ctransgenic mousexe2x80x9d of the present invention is a transgenic mouse wherein the DNA (cDNA or genomic DNA) prepared as mentioned above encoding the protein of the present invention derived from animals except mice (non-self protein) have been integrated into its endogenous locus of the mouse. The transgenic mouse expresses the non-self protein and secretes the protein into its body.
The transgenic mouse can be prepared according to the method as usually used for producing a transgenic animal (for example, see xe2x80x9cNewest Manual of Animal Cell Experimentxe2x80x9d, LIC press, Chapter 7, pp.361-408, (1990)).
Specifically, for example, embryonic stem cells (ES cells) obtained from normal mouse blastocysts are transformed with an expression vector in which the gene encoding human-derived polypeptide of the present invention (i.e. xe2x80x9chuman JTT-1 antigenxe2x80x9d) has been operably inserted. ES cells in which the gene encoding the human-derived polypeptide of the present invention has been integrated into the endogenous gene are screened by the usual method. Then, the ES cells screened are microinjected into a fertilized egg obtained from another normal mouse (blastocyst) (Proc. Natl. Acad. Sci. USA, Vol.77, No.12, pp.7380-7384 (1980); U.S. Pat. No. 4,873,191). The blastocyst is transplanted into the uterus of another normal mouse as the foster mother. Then, founder mice (progeny mice) are born from the foster mother mouse. By mating the founder mice with normal mice, heterogeneic transgenic mice are obtained. By mating the heterogeneic transgenic mice with each other, homogeneic transgenic mice are obtained according to Mendel""s laws.
xe2x80x9cKnockout mousexe2x80x9d of the present invention is a mouse wherein the endogenous gene encoding the mouse-derived protein of the present invention has been knocked out (inactivated). It can be prepared, for example, by positive-negative selection method in which homologous recombination is applied (U.S. Pat. Nos. 5,464,764; 5,487,992; 5,627,059; Proc. Natl. Acad. Sci. USA, Vol.86, pp.8932-8935 (1989); Nature, Vol.342, pp.435-438 (1989); etc.).
The xe2x80x9cantibodyxe2x80x9d of the present invention can be a polyclonal antibody (antiserum) or a monoclonal antibody, and preferably a monoclonal antibody.
Specifically, it is an antibody reactive to (against, which binds to) the above-mentioned protein or its fragment of the present invention.
The antibody of the present invention can be natural antibodies obtained by immunizing mammals such as mice, rats, hamsters, guinea pigs, and rabbits with an immunogen (antigen), such as the protein of the present invention (natural, recombinant, or synthetic ones), cells expressing the protein of the present invention, or transformants overexpressing the designed protein on the surface thereof prepared using recombinant DNA technology as described above on the cell surface. The antibody of the present invention also includes chimeric antibodies and humanized antibodies (CDR-grafted antibodies) that can be produced by recombinant DNA technology, and human antibodies that can be produced using human antibody-producing transgenic animals.
The monoclonal antibody includes those having any one isotype of IgG, IgM, IgA, IgD, or IgE. IgG or IgM is preferable.
The polyclonal antibody (antisera) or monoclonal antibody of the present invention can be produced by the known methods. Namely, a mammal, preferably, a mouse, rat, hamster, guinea pig, rabbit, cat, dog, pig, goat, horse, or cattle, or more preferably, a mouse, rat, hamster, guinea pig, or rabbit is immunized, for example, with an immunogen (antigen) mentioned above with Freund""s adjuvant, if necessary. The polyclonal antibody can be obtained from the antiserum obtained from the animal so immunized. In addition, the monoclonal antibodies are produced as follows. Hybridomas are prepared from the antibody-producing cells obtaind from the animal so immunized and myeloma cells that are not capable of producing autoantibodies. The hybridomas are cloned, and clones producing the monoclonal antibodies showing the specific affinity to the antigen used for immunizing the mammal are screened.
Specifically, the monoclonal antibody can be produced as follows. Immunizations are performed by injecting or implanting once or several times the protein of the present invention, cells expressing the protein and so on as mentioned above as an immunogen, if necessary, with Freund""s adjuvant, subcutaneously, intramuscularly, intravenously, through the footpad, or intraperitoneally into a mouse, rat, hamster, guinea pig, or rabbit, preferably a mouse, rat, or hamster (including a transgenic animal generated so as to produce antibodies derived from another animal such as the transgenic mouse producing human antibody). Usually, immunizations are performed once to four times every one to fourteen days after the first immunization. Antibody-producing cells are obtained from the mammal so immunized in about one to five days after the last immunization.
Hybridomas that secrete a monoclonal antibody can be prepared by the method of Kxc3x6hler and Milstein (Nature, Vol.256, pp.495-497 (1975)) and by its modified method. Namely, hybridomas are prepared by fusing antibody-producing cells contained in a spleen, lymph node, bone marrow, or tonsil obtained from the mammal immunized as mentioned above, preferably a spleen, with myelomas without autoantibody-producing ability, which are derived from, preferably, a mammal such as a mouse, rat, guinea pig, hamster, rabbit, or human, or more preferably, a mouse, rat, or human.
For example, mouse-derived myeloma P3/X63-AG8.653 (653), P3/NSI/1-Ag4-1 (NS-1), P3/X63-Ag8.U1 (P3U1), SP2/0-Agl4 (Sp2/0, Sp2), PAI, F0, or BW5147, rat-derived myeloma 210RCY3-Ag.2.3., or human-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11, or CEM-T15 can be used as a myeloma used for the cell fusion.
Hybridoma clones producing monoclonal antibodies can be screened by cultivating hybridomas, for example, in microtiter plates and by measuring the reactivity of the culture supernatant in the well in which hybridoma growth is observed, to the immunogen used for the immunization mentioned above, for example, by enzyme immunoassay such as RIA and ELISA.
The monoclonal antibodies can be produced from hybridomas by cultivating the hybridomas in vitro or in vivo such as in the ascites fluid of a mouse, rat, guinea pig, hamster, or rabbit, preferably a mouse or rat, more preferably mouse and isolating the antibodies from the resulting the culture supernatant or ascites fluid of a mammal.
Cultivating hybridomas in vitro can be performed depending on the property of cells to be cultured, on the object of a test study, and on the various conditions of a cultivating method, by using known nutrient media or any nutrient media derived from known basal media for growing, maintaining, and storing the hybridomas to produce monoclonal antibodies in culture supernatant.
Examples of basal media are low calcium concentration media such as Hamxe2x80x2F12 medium, MCDB153 medium, or low calcium concentration MEM medium, and high calcium concentration media such as MCDB104 medium, MEM medium, D-MEM medium, RPMI1640 medium, ASF104 medium, or RD medium. The basal media can contain, for example, sera, hormones, cytokines, and/or various inorganic or organic substances depending on the objective.
Monoclonal antibodies can be isolated and purified from the culture supernatant or ascites fluid mentioned above by saturated ammonium sulfate precipitation, euglobulin precipitation method, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52), affinity chromatography using anti-immunoglobulin column or protein A column.
The xe2x80x9cchimeric antibodyxe2x80x9d of the present invention is a monoclonal antibody prepared by genetic engineering, and specifically means a chimeric antibody such as mouse/human chimeric monoclonal antibody whose variable regions or the other regions are derived from mouse immunoglobulin and whose constant regions are derived from human immunoglobulin.
The constant region derived from human immunoglobulin has the amino acid sequence inherent in each isotype such as IgG, IgM, IgA, IgD, and IgE. The constant region of the recombinant chimeric monoclonal antibody of the present invention can be that of human immunoglobulin belonging to any isotype. Preferably, it is the constant region of human IgG.
The chimeric monoclonal antibody of the present invention can be produced, for example, as follows. Needless to say, the production method is not limited thereto.
A mouse/human chimeric monoclonal antibody can be prepared, referring to Experimental Medicine: SUPPLEMENT, Vol.1.6, No.10 (1988); and examined published Japanese patent application (JP-B) No. Hei 3-73280. Namely, it can be prepared by operably inserting CH gene (C gene encoding the constant region of H chain) obtained from the DNA encoding human immunoglobulin downstream of active VH genes (rearranged VDJ gene encoding the variable region of H chain) obtained from the DNA encoding a mouse monoclonal antibody isolated from the hybridoma producing the mouse monoclonal antibody, and CL gene (C gene encoding the constant region of L chain) obtained from the DNA encoding human immunoglobulin downstream of active VL genes (rearranged VJ gene encoding the variable region of L chain) obtained from the DNA encoding the mouse monoclonal antibody isolated from the hybridoma, into the same or different vectors so as for them to be expressed, following by transforming host cells with the expression vector, and then by cultivating the transformants.
Specifically, DNAs are first extracted from mouse monoclonal antibody-producing hybridomas by the usual method, digested with appropriate restriction enzymes (for example, EcoRI and HindIII), electrophoresed (using, for example, 0.7% agarose gel), and analyzed by Southern blotting. After an electrophoresed gel is stained, for example, with ethidium bromide and photographed, the gel is given with marker positions, washed twice with water, and soaked in 0.25 M HCl for 15 minutes. Then, the gel is soaked in 0.4 N NaOH solution for 10 minutes with gently stirring. The DNAs are transferred to a filter for 4 hours by the usual method. The filter is recovered and washed twice with 2xc3x97SSC. After the filter is sufficiently dried, it is baked at 75xc2x0 C. for 3 hours. After baking, the filter is treated with 0.1xc3x97SSC/0.1% SDS at 65xc2x0 C. for 30 minutes. Then, it is soaked in 3xc3x97SSC/0.1% SDS. The filter obtained is treated with prehybridization solution in a plastic bag at 65xc2x0 C. for 3 to 4 hours.
Next, 32P-labeled probe DNA and hybridization solution are added to the bag and reacted at 65xc2x0 C. about 12 hours. After hybridization, the filter is washed under appropriate salt concentration, reaction temperature, and time (for example, 2xc3x97SSC-0.1% SDS, room temperature, 10 minutes). The filter is put into a plastic bag with a little 2xc3x97SSC, and subjected to autoradiography after the bag is sealed.
Rearranged VDJ gene and VJ gene encoding H chain and L chain of a mouse monoclonal antibody are identified by Southern blotting mentioned above. The region comprising the identified DNA fragment is fractioned by sucrose density gradient centrifugation and inserted into a phage vector (for example, Charon 4A, Charon 28, xcexEMBL3, xcexEMBL4, etc.). E. coli (for example, LE392, NM539, etc.) is transformed with the phage vector to generate a genomic library. The genomic library is screened by plaque hybridization such as Benton-Davis method (Science, Vol.196, pp.180-182 (1977)) using appropriate probes (H chain J gene, L chain (xcexa) J gene, etc.) to obtain positive clones comprising rearranged VDJ gene or VJ gene. By making the restriction map and determining the nucleotide sequence of the clones obtained, it is confirmed that genes comprising the desired, rearranged VH (VDJ) gene or VL (VJ) gene are obtained.
Separately, human CH gene and human CL gene used for chimerization are isolated. For example, when a chimeric antibody with human IgG1 is produced, Cxcex31 gene as a CH gene, and Cxcexa gene as a CL gene, are isolated. These genes can be isolated from human genomic library with mouse Cxcex31 gene and mouse Cxcexa gene, corresponding to human Cxcex31 gene and human Cxcexa gene, respectively, as probes, taking advantage of high homology between the nucleotide sequences of mouse immunoglobulin gene and that of human immunoglobulin gene.
Specifically, DNA fragments comprising human Cxcexa gene and an enhancer region are isolated from human xcex Charon 4A HaeIII-AluI genomic library (Cell, Vol.15, pp.1157-1174 (1978)), for example, with a 3 kb HindIII-BamHI fragment of clone Ig146 (Proc. Natl. Acad. Sci. USA, Vol.75, pp.4709-4713 (1978)) and a 6.8 kb EcoRI fragment of clone MEP10 (Proc. Natl. Acad. Sci. USA, Vol.78, pp.474-478 (1981)) as probes. In addition, for example, after human fetal hepatocyte DNA is digested with HindIII and fractioned by agarose gel electrophoresis, a 5.9 kb fragment is inserted into xcex788 and then human Cxcex31 gene is isolated with the probes mentioned above.
Using mouse VH gene, mouse VL gene, human CH gene, and human CL gene so obtained, and taking promoter region and enhancer region into consideration, human CH gene is inserted downstream mouse VH gene and human CL gene is inserted downstream mouse VL gene into an expression vector such as pSV2gpt or pSV2neo with appropriate restriction enzymes and DNA ligase by the usual method. In this case, chimeric genes of mouse VH gene/human CH gene and mouse VL gene/human CL gene can be respectively inserted in the same expression vector or in different expression vectors.
Chimeric gene-inserted expression vector(s) thus prepared are introduced into myelomas that do not produce antibodies, for example, P3X63.Ag8.653 cells or SP210 cells by protoplast fusion method, DEAE-dextran method, calcium phosphate method, or electroporation method. The transformants are screened by cultivating in media containing a drug corresponding to the drug resistance gene inserted into the expression vector and, then, cells producing desired chimeric monoclonal antibodies are obtained.
Desired chimeric monoclonal antibodies are obtained from the culture supernatant of antibody-producing cells thus screened.
The xe2x80x9chumanized antibody (CDR-grafted antibody)xe2x80x9d of the present invention is a monoclonal antibody prepared by genetic engineering and specifically means a humanized monoclonal antibody wherein a portion or the whole of the complementarity determining regions of the hypervariable region are derived from the complementarity determining regions of the hypervariable region from a mouse monoclonal antibody, the framework regions of the variable region are derived from the framework regions of the variable region from human immunoglobulin, and the constant region is derived from human a constant region from immunoglobulin.
The complementarity determining regions of the hypervariable region exists in the hypervariable region in the variable region of an antibody and means three regions which directly and complementary binds to an antigen (complementarity-determining residues, CDR1, CDR2, and CDR3). The framework regions of the variable region means four comparatively conserved regions lying upstream, downstream or between the three complementarity determining regions (framework region, FR1, FR2, FR3, and FR4).
In other words, a humanized monoclonal antibody means that in which the whole region except a portion or the whole of the complementarity determining regions of the hypervariable region of a nonhuman mammal-derived monoclonal antibody have been replaced with their corresponding regions derived from human immunoglobulin.
The constant region derived from human immunoglobulin has the amino acid sequence inherent in each isotype such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, and IgE. The constant region of a humanized monoclonal antibody in the present invention can be that from human immunoglobulin belonging to any isotype. Preferably, it is the constant region of human IgG. The framework regions of the constant region derived from human immunoglobulin are not particularly limited.
The humanized monoclonal antibody of the present invention can be produced, for example, as follows. Needless to say, the production method is not limited thereto.
For example, a recombinant humanized monoclonal antibody derived from mouse monoclonal antibody can be prepared by genetic engineering, referring to unexamined Japanese patent publication (JP-WA) No. Hei 4-506458 and unexamined Japanese patent publication (JP-A) No. Sho 62-296890. Namely, at least one mouse H chain CDR gene and at least one mouse L chain CDR gene corresponding to the mouse H chain CDR gene are isolated from hybridomas producing mouse monoclonal antibody, and human H chain gene encoding the whole regions except human H chain CDR corresponding to mouse H chain CDR mentioned above and human L chain gene encoding the whole region except human L chain CDR correspond to mouse L chain CDR mentioned above are isolated from human immunoglobulin genes.
The mouse H chain CDR gene(s) and the human H chain gene(s) so isolated are operably inserted into an appropriate vector so that they can be expressed. Similarly, the mouse L chain CDR gene(s) and the human L chain gene(s) are operably inserted into another appropriate vector so that they can be expressed. Alternatively, the mouse H chain CDR gene(s)/human H chain gene(s) and mouse L chain CDR gene(s)/human L chain gene(s) can be operably inserted into the same expression vector so that they can be expressed. Host cells are transformed with the expression vector thus prepared to obtain transformants producing humanized monoclonal antibody. By cultivating the transformants, desired humanized monoclonal antibody is obtained from culture supernatant.
The xe2x80x9chuman monoclonal antibodyxe2x80x9d of the present invention is immunoglobulin in which the entire regions comprising the variable and constant region of H chain, and the variable and constant region of L chain constituting immunoglobulin are derived from the gene encoding human immunoglobulin. The human antibody can be produced in the same way as the production method of polyclonal or monoclonal antibodies mentioned above by immunizing, with an antigen, a transgenic animal which for example, at least human immunoglobulin gene(s) have been integrated into the locus of a non-human mammal such as a mouse by the usual method. For example, a transgenic mouse producing human antibodies is prepared by the methods described in Nature Genetics, Vol.15, pp.146-156 (1997); Nature Genetics, Vol.7, pp.13-21 (1994); JP-WA Nos. Hei4-504365, International patent publication No. WO94/25585; Nikkei Science, No.6, pp.40-50 (1995); Nature, Vol.368, pp.856-859 (1994); and JP-WA No. Hei 6-500233.
The xe2x80x9cportion of an antibodyxe2x80x9d used in the present invention means a partial region of the antibody, preferably monoclonal antibody of the present invention as mentioned above, and specifically, means F(abxe2x80x2)2, Fabxe2x80x2, Fab, Fv (variable fragment of antbody), sFv, dsFv (disulfide stabilized Fv), or dAb (single domain antibody) (Exp. opin. Ther. Patents, Vol.6, No.5, pp.441-456 (1996)).
xe2x80x9cF(abxe2x80x2)2xe2x80x9d and xe2x80x9cFabxe2x80x2xe2x80x9d can be produced by treating immunoglobulin (monoclonal antibody) with a protease such as pepsin and papain, and means an antibody fragment generated by digesting immunoglobulin near the disulfide bonds existing between the hinge regions in each of the two H chains. For example, papain cleaves IgG upstream of the disulfide bonds existing between the hinge regions in each of the two H chains to generate two homologous antibody fragments in which an L chain composed of VL (L chain variable region) and CL (L chain constant region), and an H chain fragment composed of VH (H chain variable region) and CHxcex31 (xcex31 region in the constant region of H chain) are connected at their C terminal regions through a disulfide bond. Each of such two homologous antibody fragments is called Fabxe2x80x2. Pepsin also cleaves IgG downstream of the disulfide bonds existing between the hinge regions in each of the two H chains to generate an antibody fragment slightly larger than the fragment in which the two above-mentioned Fabxe2x80x2 are connected at the hinge region. This antibody fragment is called F(abxe2x80x2)2.
The xe2x80x9cpharmaceutical compositionxe2x80x9d of the present invention comprises any one of the protein, protein fragment, fusion protein antibody, or portion of an antibody of the present invention as defined above; and a pharmaceutically acceptable carrier.
The xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d includes a excipieut, a diluent, an expander, a decomposition agent, a stabilizer, a preservative, a buffer, an emulsifier, an aromatic, a colorant, a sweetener, a viscosity increasing agent, a flavor, a solubility increasing agent, or other additives. Using one or more of such carriers, a pharmaceutical composition can be fomulated into tablets, pills, powders, granules, injections, solutions, capsules, troches, elixirs, suspensions, emulsions, or syrups. The pharmaceutical composition can be administered orally or parenterally. Other forms for parenteral administration include a solution for external application, suppository for rectal administration, and pessary, prescribed by the usual method, which comprises one or more active ingredient.
The dosage can vary depending on the age, sex, weight, and symptom of a patient, effect of treatment, administration route, period of treatment, or the kind of active ingredient (polypeptide or antibody mentioned above) contained in the pharmaceutical composition. Usually, the pharmaceutical composition can be administered to an adult in a dose of 10 xcexcg to 1000 mg (or 10 xcexcg to 500 mg) per one administration. Depending on various conditions, the dosage less than that mentioned above may be sufficient in some cases, and the dosage more than that mentioned above may be necessary in other cases.
In particular, the injection. can be produced by dissolving or suspending the antibody in a non-toxic, pharmaceutically acceptable carrier such as physiological saline or commercially available distilled water for injection with adjusting a concentration to 0.1 xcexcg antibody/ml carrier to 10 mg antibody/ml carrier. The injection thus produced can be administered to a human patient in need of treatment in a dose of 1 xcexcg to 100 mg/kg body weight, preferably 50 xcexcg to 50 mg/kg body weight once or more times a day. Examples of administration route are medically appropriate administration routes such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, or intraperitoneal injection, preferably intravenous injection.
The injection can also be prepared into a non-aqueous diluent (for example, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and alcohol such as ethanol), suspension, or emulsion.
The injection can be sterilized by filtration with a bacteria-non-penetrated filter, by mixing bacteriocide, or by irradiation. The injection can be produced in the form that is prepared upon use. Namely, it is freeze-dried to be a sterile solid composition, and can be dissolved in sterile distilled water for injection or another solvent before use.
The pharmaceutical composition of the present invention can be used to treat or prevent arteriosclerosis and restenosis after the treatment of artery occlusion, such as PTCA.